Important functions in the field of drug discovery, known as pharmacokinetics and pharmacodynamics (PK and PD), involve the quantification of one or more test compounds in a biological sample such as plasma, urine, cerebrospinal fluid (CSF), or similar. In the discovery or early development phases of creating new pharmacological compounds, PK and PD experiments typically involve dosing a test animal and monitoring the level of the test compound and/or its metabolites in various biological fluids or tissues as a function of time.
A common method for the quantification of test compounds in biological samples involves the use of a radioisotope of the test compound. The concentration of the test compound can be measured by tracking the amount of radioactivity present in each sample. However, often a radioisotope of the test compound of interest is not available. In such cases mass spectrometry (MS) is commonly used as the analytical method for the quantification of test compounds. MS is a powerful technique that can be used to selectively and quantitatively detect one or more analytes of interest in a complex mixture based on the molecular mass of those analytes.
While MS is a very powerful technique that is commonly used in many areas of the drug discovery process, it has certain limitations. One requirement for MS is that the analytes, typically in liquid phase, to be detected need to be ionized and fully desolvated to enter the MS inlet and reach the detector. There are several commonly used methods for ionizing and desolvating samples including electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photo ionization (APPI). These methods are often collectively called atmospheric pressure ionization (API). In all API methods, the samples are ionized through the application of a strong electric field that is typically many thousands of volts in magnitude. Often a heated inert gas, such as nitrogen, is applied to evaporate the solvent associated with the sample resulting in an analyte that is ionized and is in the gas phase.
Unfortunately, this approach of sample ionization does not work well for samples that contain high levels of ions, such as salts and/or buffers. In samples that contain high levels of ions, a phenomenon known as ion suppression occurs, in which the bulk of the ionization occurs within the salt or buffer molecules and little or no analyte of interest is actually ionized. The result is that the sensitivity of the MS technique is dramatically reduced when such samples are analyzed. Furthermore, most salts and buffer are not volatile and, when desolvated in an API MS instrument, tend to precipitate in the source region of the instrument and often cause a further decline in instrument performance.
To overcome the problems of ion suppression samples to be analyzed are generally purified and/or fractionated prior to MS analysis by some form of chromatography. In general, there are two major forms of sample preparation in MS analysis that can be described as on-line techniques and decoupled techniques. In decoupled techniques, the samples are purified off-line in a separate step such as solid-phase extraction (SPE) or liquid-liquid extraction (LLE). Often, many samples are purified together in parallel to provide an increase in throughput. On-line techniques are also sometimes described as “hyphenated” methods and involve coupling some form of chromatography system, such as liquid chromatography (LC) directly with the mass spectrometer such that the eluate from the chromatography system in analyzed by the MS. Often, high-pressure liquid chromatography (HPLC) is interfaced with the MS in a technique known as HPLC-MS.
Although HPLC-MS is an immensely powerful technique for biological analysis, it is limited by a relatively slow throughput, which is typically on the order of several minutes per sample. In cases where a large number of samples must be analyzed, the bioanalysis using HPLC-MS can create a significant bottleneck. While various methods have been described for increasing the throughput of the HPLC-MS process, including multiplexing of a plurality of HPLC system to a single MS or decreasing the run times of HPLC systems through the use of smaller particles or a ballistic gradient, most commercially available systems still require more than 1 minute per sample. The RAPIDFIRE® system by BioTrove, Inc. of Woburn, Mass. uses a miniaturized and fully automated on-line solid-phase extraction system coupled to a mass spectrometer (SPE-MS) and has been shown to facilitate MS analysis at throughputs up to 6 seconds per sample for a wide range of applications including high-throughput screening and in vitro ADME (related RF patents should be referenced).
PK and PD analysis, particularly from plasma samples, create a significant challenge for an SPE-MS system due to the limited sample fractionation afforded by the SPE step. While very effective at removing salts and buffers, the SPE method does little or no fractionation of other compounds in the sample. In complex biological samples such as plasma, this could translate to a very large number of compounds at high concentrations that can cause significant ion suppression.
In the case of plasma samples there are typically two major issues that must be overcome. The first is a high concentration of proteins, notably serum albumin, which can be found at concentrations as high as 20 mg/mL. At these concentrations proteins tend to precipitate in organic solvents, such as acetonitrile and methyl alcohol, which are commonly used to elute the analytes from the SPE columns creating the physical challenge of clogging as well as ion suppression. Fortunately, removing proteins from samples can be achieved very quickly and simply with a variety of techniques which are well known to those skilled in the practice.
A bigger challenge for the analysis of PK/PD samples using SPE-MS techniques is the presence of millimolar concentrations of a wide range of phospholipids in plasma. These phospholipids are typically retained on the standard reversed-phase solid-phase extraction systems that are routinely used in most SPE-MS applications. The result is that the phospholipids co-purify with the analytes of interest and generally result in significant ion suppression. Typically, due to the effects of phospholipid ion-suppression, the lower limit of quantification (LLQ) of an analyte of interest for a SPE-MS method does not compare with that of an HPLC-MS method where the analyte of interest is fractionated and separated from the phospholipids.
Accordingly, there is a need for systems and methods the enable the use of SPE-MS methods for high-throughput PK/PD analysis of plasma samples while achieving limits of quantification that are comparable to HPLC-MS methods.